Renal-protective effects of Chinese medicinal herbs and compounds for diabetic kidney disease in animal models: protocol for systematic review and meta-analysis

Background Diabetic kidney disease (DKD) is a common and severe complication of diabetes that can lead to end-stage renal disease with no cure. The first-line drugs recommended by clinical guidelines fail to achieve satisfactory effects for people with DKD. A Chinese herbal medicine Tangshen Qushi Formula (TQF) shows preliminary efficacy and safety in preserving renal function for people with DKD, but the effects on comprehensive renal outcomes remain unclear. We will conduct a systematic review and meta-analysis to evaluate the effects of TQF herbs and their compounds identified from ultra-high performance liquid chromatography-MS/MS in diabetic animal models with renal outcomes. Methods This protocol complies with the guideline Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols. We will include studies investigating the effects of TQF herbs and compounds on diabetic rats or mice with renal outcomes. Six electronic databases will be searched from their inception to February 2023. Quality assessment will be conducted using SYRCLE’s risk of bias tool. Standardized or weighted mean differences will be estimated for renal outcomes (creatinine, urea, proteinuria, histological changes, oxidative stress, inflammation, and kidney fibrosis). Data will be pooled using random-effects models. Heterogeneity across studies will be expressed as I2. Sensitivity analyses will explore treatment effects in adjusted models and within subgroups. Funnel plots and Egger’s test will be used to explore publication bias. Discussion The results of this review will provide valuable insights into the potential effects of TQF in managing DKD. The limitation is that the included studies will be animal studies from specific databases, and the interpretation of the findings must be cautious. Systematic review registration PROSPERO CRD42023432895. Registered on 19 July 2023 (https://www.crd.york.ac.uk/PROSPERO/#recordDetails). Supplementary Information The online version contains supplementary material available at 10.1186/s13643-023-02446-4.

place of origin were provided in the S1 Table .The purity/contaminant tests showed that the TQF granules were free of heavy metals or other undesirable contaminants, which complied with the pharmacopeial criteria (2020 Edition (34)).The stability/consistency tests are being performed to indicate whether the product is stable/consistent from the start till the end of the clinical study.No equivalence test was needed since a single batch of TQF granules would be used during the clinical study.The  The extraction procedure of the TQF product was: firstly, do two rounds of decocting for the mixture of seven raw botanical drugs with water.Secondly, the decoctions from the previous step are merged, then filtered and concentrated into a transparent paste.Next, maltodextrin was added to the paste till it was dissolved and then spray-dried.The mixture was made into 1000 grams of granules.In the end, the granules were packaged into packets of 7.5 grams.A single batch was used for this clinical study.

Preparation of placebo
The TQF placebo was produced by the same manufacturer using a concentrate of caramel pigments, gardenia yellow pigment, sunset yellow pigment, and dextrin with the proportion of 1.5: 1: 1: 0.8.Other components were sucrose octaacetate (3%), suspending agent (dextrin and titanium dioxide, 0.8%), and lactose (10%).The placebo was made in granules and packaged into packets of 7.5 grams.According to the 2020 Chinese pharmacopeia, the components in the placebo are food-grade at this dosage and have no known side effects with oral consumption.The blinding test of the TQF granules and placebo showed the package and weight were identical, and the appearance, color, smell, and taste were similar.Critical information on chemicals, manufacturing, and control of TQF and TQF placebo is being conducted by the pharmaceutical team of GPHCM.The chemical composition of TQF is validated using high-performance liquid chromatography/mass spectrometry.

Chemical analysis and quality control
Ultra-high performance liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS) analysis was adopted for the TQF granules.The supernatant of granules was ultrasonically extracted with 50% methanol.UHPLC analysis was performed using a Thermo UltiMate 3000 UHPLC instrument (Waltham, MA, USA).The analysis employed an ACQUITY UPLC® HSS T3 C18 column (2.1 mm × 100 mm, 1.8 μm, Waters, USA).The mobile phase comprised acetonitrile (A) and water with 0.1% formic acid (B).The flow rate was maintained at 200 μL/min.A gradient elution system was employed as follows: S2 The chemicals contained in TQF were assessed by high-performance liquid chromatography (HPLC) spectrometry.HPLC analysis was performed using a Waters 2695 instrument (Waltham, MA, USA).The parameters were as follows: Column: Xbridge® C18, 4.6 mm×250 mm, 5 μm (Waters, USA); Mobile phase: Acetonitrile (A), water with 0.1% formic acid (B).A gradient elution system was employed: injection volume of 5 μL was used for each sample.Mass Spectrometry: Mass spectrum acquisition was carried out using a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific, San Jose, USA) equipped with heated electrospray ionization (HESI) operating in the negative ionization mode.The main parameters optimized for mass spectrometry were set as follows: was imported into Compound Discoverer 3.0 software, and the possible molecular formulas were fitted through the extracted molecular ion chromatographic peaks.The results were then matched with the mz Vault and mz Cloud databases.Xcalibur 2.1 software was used to further analyze and identify the compounds by comparing them with relevant literature and standard substances based on the accurate m/z values of precursor and fragment ions, retention time, and the information of cleavage pattern.Thirty-eight compounds were identified from UHPLC-MS/MS analysis of TQF (Fig 2 in the manuscript).The details of the compounds and their bioactivities are listed in the S2 Table.
components of TQF in scientific, species, pharmaceutical, and Chinese Pin Yin names, proportion, and place of origin

Table Compounds from UHPLC-MS/MS analysis of TQF and their bioactivities
* Compared with reference compounds